Low temperature is a critical environmental factor that affects growth and survival of many plant species and limits their geographical distribution. Overwintering plants are able to increase their freezing tolerance when exposed to low but non-freezing temperatures, a process known as cold acclimation. During cold acclimation, several cellular and physiological changes occur, including alterations in gene expression. Cold-regulated (COR) genes are highly induced during cold acclimation and are involved in the acquisition of freezing tolerance (Thomashow, 1998). Expression of COR genes is activated by C-repeat binding factors (CBFs), the best-characterized transcription factors related to acquiring freezing tolerance (Jaglo-Ottosen et al., 1998; Liu et al., 1998). Since CBFs are plant specific (Riechmann and Meyerowitz, 1998; Riechmann et al., 2000; Sakuma et al., 2002), it is thought that the CBF signal transduction pathway is conserved only among plants. However, low temperature affects growth and development in diverse organisms. Therefore, it is reasonable to speculate that there are mechanisms for adaptation to low temperature that are conserved throughout evolution, as is known for heat-shock responses. To date, it is not well understood whether there are conserved responses to low temperature within prokaryotes and eukaryotes.

Cold acclimation is also observed in bacteria and has been extensively studied in Escherichia coli. The major cold shock protein (CSP) in E. coli, CspA, is predominantly induced after exposure to low temperature and accumulates to up to 10% of the total protein in the cell (Goldstein et al., 1990). E. coli contains nine cspA family genes (cspA to cspI) and four of them (cspA, cspB, cspG, and cspI) are induced by cold shock (Yamanaka et al., 1998; Wang et al., 1999). The cspC and cspE genes are expressed constitutively and involved in the chromosome partitioning (Yamanaka et al., 1994). Expression of cspD is induced at the stationary phase or upon nutrient starvation. Overexpression of cspD results in a lethal phenotype in E. coli (Yamanaka and Inouye, 1997; Xia et al., 2001). The E. coli cspA, cspB, cspG, and cspE quadruple deletion mutant shows a cold-sensitive phenotype, which can be complemented by overexpression of any one of the CSPs except CspD (Xia et al., 2001). CSPs unwind nucleic acid duplexes in vitro and in vivo (Jiang et al., 1997; Bae et al., 2000; Phadtare et al., 2002). RNA molecules tend to form stable secondary structures at low temperatures, which may impede RNA function such as in translation and transcription. Therefore, it has been suggested that CSPs act as RNA chaperones to destabilize RNA secondary structures, enabling efficient translation at low temperature (Jiang et al., 1997; Phadtare et al., 2002). In addition, CspA, CspC, and CspE are transcription antiterminators, which regulate expression of a set of cold-inducible genes (Bae et al., 2000). Bacterial CSPs are composed of a single nucleic acid binding domain (of about 70 amino acid residues) called the cold shock domain (CSD). The CSD consists of a five-stranded β-barrel containing two consensus RNA binding motifs (RNP-1 and RNP-2), which contribute to single-stranded DNA/RNA binding (Schroder et al., 1995; Hillier et al., 1998; Wang et al., 2000).

In animals, the homologous genes to bacterial CSPs have been identified, and have been shown to contain an N-terminal CSD and C-terminal auxiliary domains. The structure of the auxiliary domains varies significantly in different organisms. The vertebrate Y-box binding (YB) proteins have a highly conserved CSD, which shares about 40% amino acid sequence identity with bacterial CSPs (Wistow, 1990; Wolffe et al., 1992). YB proteins contain an N-terminal Ala and Pro rich domain (A/P domain) followed by a CSD and C-terminal alternating clusters of basic and acidic amino acid residues (B/A repeat; Wolffe, 1994; Graumann and Marahiel, 1998). The C-terminal B/A repeat was proposed to have DNA and RNA binding activity and the ability to bind other proteins (Evdokimova and Ovchinnikov, 1999). YB-1, the best-characterized YB protein, is a multifunctional protein that is involved in the regulation of transcription and translation, drug resistance, cell proliferation, and stress adaptation (Kohno et al., 2003). After a temperature downshift, no increase in cell numbers was observed for YB-1-depleted chicken cells, indicating an essential function for YB-1 in cell proliferation under cold conditions (Matsumoto et al., 2005). YB-1 binds to DNA and RNA and shows concentration-dependent melting and annealing activities (Matsumoto and Wolffe, 1998; Skabkin et al., 2001). These activities are probably necessary for the pleiotropic functions of YB-1. Another example of a eukaryotic CSD protein is LIN-28, which was originally identified as an essential regulator of larval development in C. elegans (Moss et al., 1997). LIN-28 comprises a single CSD and two CCHC retroviral-like zinc fingers at the C-terminus. Mammalian LIN-28 is a translational enhancer of IGF-2, which is essential for the growth and differentiation of muscle tissue (Polesskaya et al., 2007). In addition, it has been demonstrated that LIN-28 plays an important role in reprogramming human somatic cells into pluripotent stem cells (Yu et al., 2007; Liao et al., 2008). LIN-28 post-transcriptionally inhibits the biogenesis of let-7 miRNA, which is a regulator of cell growth and differentiation in embryonic cells (Roush and Slack, 2008; Viswanathan et al., 2008). Collectively, these studies suggest that animal CSD proteins play important roles in a variety of biological processes, not only stress adaptation.

Cold shock domain proteins are widespread among lower and higher plants including monocots and dicots (Karlson and Imai, 2003). Plant CSD proteins so far characterized typically contain a large glycine-rich region interspersed with CCHC zinc fingers at the C-terminus (Karlson et al., 2002), a domain structure, and arrangement resembling that of LIN-28. We thus conducted a database search using Plant GDB (http://www.plantgdb.org/) to identify CSD proteins in plant species whose genome sequences are available (Figure 1). All known genome sequences contain at least two CSD proteins that are composed of a CSD, glycine-rich regions and CCHC zinc fingers. The sequence information also indicates that genomic sequences for CSD proteins commonly contain no introns. The number of gene family members in each species ranges from two (Oryza sativa, Zea mays, Sorghum bicolor, and Vitis vinifera) to seven (Glycine max). In addition, individual species have both long and short versions of CSD proteins. In monocots, CSD proteins contain two or four CCHC zinc fingers, whereas the number of CCHC zinc fingers is one, two, four, five, or seven in dicots. The glycine-rich and CCHC zinc finger regions are probably involved in binding of nucleic acids and other proteins (Karlson et al., 2002; Nakaminami et al., 2006; Sasaki et al., 2007). Since all plant CSD proteins contain a highly conserved CSD, it is speculated that the diverse combinations of CCHC zinc fingers are necessary for binding to specific nucleic acids and proteins.

Whereas a considerable amount of research has been carried out to characterize CSD proteins in bacteria and animals, little is known about their functions in plants. The first functionally characterized plant CSD protein was the wheat CSP (WCSP1; Karlson et al., 2002; Table 1). WCSP1 contains a glycine-rich region interspersed with three C-terminal CCHC zinc fingers. WCSP1 mRNA is up-regulated in response to cold and the corresponding protein is substantially accumulated in crown tissue during prolonged cold acclimation. Transcript levels of WCSP1 are not modulated by other environmental stresses such as salt, drought and heat, or treatment with abscisic acid (Karlson et al., 2002), suggesting that the function of WCSP1 is specific to cold adaptation. WCSP1 binds to DNA and RNA and melts double-stranded nucleic acids in vitro and in vivo (Karlson et al., 2002; Nakaminami et al., 2005, 2006). In addition, WCSP1 complements a cold-sensitive phenotype of the E. coli csp mutant (Nakaminami et al., 2006). These data suggest that WCSP1 shares a conserved function with E. coli CSPs and is involved in the regulation during cold acclimation.

Rice has two CSD proteins [OsCSP1 (Os02g0121100) and OsCSP2 (Os08g0129200)], which exhibit nucleic acid binding activity and complement the cold sensitivity of the E. coli csp mutant (Chaikam and Karlson, 2008). Expression of OsCSPs was slightly increased in shoot and root tissues by short term low temperature treatment (Chaikam and Karlson, 2008; Table 1). However, OsCSP protein levels were not increased in crown tissue during 10 days of low temperature treatment (Chaikam and Karlson, 2008). These data are in great contrast to the observed expression characteristics for WCSP1. Tissue specific expression patterns of OsCSPs revealed that OsCSP proteins are highly accumulated in the developing panicle, flower, and seed (Chaikam and Karlson, 2008). Thus, the functions of OsCSPs may be more associated with developmental processes than with cold tolerance.

In Arabidopsis, four CSD proteins (AtCSP1–AtCSP4) were identified and functional analyses of AtCSPs have been performed with overexpression lines and mutants (Table 1). An AtCSP3 (At2g17870) knock-out mutant (atcsp3-2) was more sensitive to freezing than was wild-type under both non-acclimated and cold-acclimated conditions (Kim et al., 2009). Overexpression of AtCSP3 confers increased freezing tolerance in Arabidopsis without obvious developmental defects (Kim et al., 2009). AtCSP3 does not affect the expression of CBFs and COR genes, but it regulates the expression of stress-related genes whose roles in freezing tolerance are unknown (Kim et al., 2009). Interestingly, several genes down-regulated in atcsp3-2 are known to be up-regulated in the ada2b-1 mutant, which is more freezing tolerant than wild-type without up-regulation of COR gene expression (Vlachonasios et al., 2003). Since ADA2b is a component of histone acetyltransferase complexes, it is speculated that there is crosstalk between AtCSP3 and histone modification. It has been demonstrated that AtCSP2 (AtGRP2/CSDP2; At4g38680) transcript is highly expressed in meristematic and developing tissues (Fusaro et al., 2007; Sasaki et al., 2007; Nakaminami et al., 2009). Consistent with its expression patterns, functional analyses using RNAi knock-down transgenic plants indicated that AtCSP2 negatively regulates flowering time, and positively regulates seed/embryo development (Fusaro et al., 2007). Recently, it was demonstrated that AtCSP4 (AtGRP2b; At2g21060), the closest paralog of AtCSP2, also plays an important role in development. Overexpression of AtCSP4 resulted in reduced silique length and embryo lethality (Yang and Karlson, 2011). Expression of several MADS-box and endosperm development genes is altered in the AtCSP4-overexpressing line during floral and silique development. Park et al. (2009) reported that overexpression of AtCSP1 (CSDP1; At4g36020) delays seed germination under dehydration or salt stress conditions, whereas overexpression of AtCSP2 accelerated seed germination under salt stress conditions. Although overexpression of AtCSP1 or AtCSP2 did not enhance freezing tolerance in Arabidopsis, they each complement the freezing-sensitive phenotype of grp7, which is a mutant of glycine-rich RNA binding protein 7 (GRP7) with RNA chaperone activity (Kim et al., 2007, 2008). These functional studies of plant CSD proteins reveal that the expressions of multiple CSD proteins are differentially regulated by developmental and stress cues (Table 1). Furthermore, plant CSD proteins commonly exhibit RNA chaperone activity and function as regulatory proteins.

Our understanding of plant CSD proteins has progressed significantly in recent years. The evolutionarily conserved structures and biochemical activities of CSD proteins suggest that these proteins are indispensable for cold adaptation in both prokaryotes and eukaryotes. In addition, regulatory functions of CSD proteins extend to developmental processes in both animals and plants. Whereas information regarding the biological functions of plant CSD proteins is accumulating, the cellular function of plant CSD proteins still remains to be elucidated. Whether or not CSD proteins have specific target mRNAs in plant cells needs to be addressed. In Chlamydomonas reinhardtii, the NAB1 CSD protein stabilizes the mRNA of LHCBM (major light-harvesting complex of photosynthesis II) and represses its translation at the pre-initiation stage (Mussgnug et al., 2005). NAB1-like proteins have not been identified in higher plants. However, RNA stabilization and translational repression have been described for animal CSD proteins such as YB-1 and FRGY2 (Matsumoto et al., 1996; Evdokimova et al., 2001). Conserved post-transcriptional regulation mechanisms may exist for NAB1 and animal CSD proteins. Further studies are necessary to investigate possible CSD-target mRNAs in higher plants. These investigations could reveal novel mechanisms of gene regulation through CSD proteins.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.